Method for SNP (single nucleotide polymorphism) typing

ABSTRACT

The present invention enables typing nearly hundreds of thousands of SNP sites using remarkably a small amount of genomic DNA. That is, the method for SNP typing of the present invention comprises the steps of simultaneously amplifying a plurality of nucleotide sequences comprising at least one or more sites of single nucleotide polymorphism (SNP) using genomic DNA and a plurality of primer pairs; and typing for distinguishing nucleotides of SNP sites contained in the plurality of nucleotide sequences amplified by the above amplification step, using the amplified nucleotide sequences.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for single nucleotidepolymorphism (so-called “SNP”) typing which is used for identifyingpolymorphism of a site of SNP in genomic DNA.

BACKGROUND OF THE INVENTION

[0002] Just as human appearances vary widely, their 3 billion geneticcodes differ at numerous sites when compared between individuals. Suchdifferences in genetic codes are called polymorphisms. A singlenucleotide polymorphism (hereinafter referred to as “SNP”) is known as atypical polymorphism.

[0003] SNP, single nucleotide polymorphism, means a single basedifference among a plurality of individuals. SNPs are classified intocSNP (coding SNP) and gSNP (genome SNP) according to their location.cSNP further includes sSNP (silent SNP), rSNP (regulatory SNP) and iSNP(intron SNP). In this specification, a site at which SNP occurs ingenomic DNA, that is, a certain nucleotide at which SNP occurs isreferred to as “single nucleotide polymorphism site” or “SNP site.”

[0004] Three to ten million SNP sites are thought to exist in the humangenome. It is thought that some of these SNPs affect the control ofexpression or functions of proteins, and some involve individualdifferences in body compositions and susceptibility to a disease. Thatis, made to order medical care can be given according to an individual'sbody composition by obtaining information on SNPs. Accordingly, SNPs areincreasingly discovered and identified, and many SNPs have already beenreported.

[0005] The next task is to analyze information about how each SNPaffects responsiveness to drugs and diseases, body composition or thelike. To accomplish the task, SNP typing (a process to discriminatenucleotides of SNP sites) must be done to know how each individual'sSNPs are. Such SNP typing would be a large-scale analytical process,such that SNP typing is performed for several hundreds of thousands ofSNP sites per individual.

[0006] Examples of SNP typing are those using genomic DNA, including aTaqMan PCR method, an invader assay, a SniPer method, a MALDI-TOF-MASSmethod, a DNA chip method and the like. All of these methods require atleast several tens of nanograms of genomic DNA to genotype one SNP site.When several hundreds of thousands of SNP sites are genotyped, severalmg of genomic DNA is required per individual.

[0007] Nearly 11 of blood must be collected from an individual to obtainseveral mg of genomic DNA, however, it is actually impossible to obtaingenomic DNA in such a volume from an individual. That is, typing ofhundreds of thousands of SNP sites per individual cannot be performed atone time.

SUMMARY OF THE INVENTION

[0008] We have completed the present invention under thesecircumstances. The purpose of the present invention is to provide amethod for SNP typing which can genotype hundreds of thousands of SNPsites using a remarkably small amount of genomic DNA.

[0009] A method for SNP typing according to the present invention, whichhas achieved the above purposes, comprises the steps of simultaneouslyamplifying a plurality of nucleotide sequences comprising at least oneor more SNP sites using genomic DNA and a plurality of primer pairs; andtyping to discriminate nucleotides of SNP sites contained in theplurality of the nucleotide sequences amplified by the aboveamplification step.

[0010] In the method for SNP typing according to the present invention,a polymerase chain reaction using a hot start method is preferably usedin the above amplification step.

[0011] Further in the method for SNP typing according to the presentinvention, 50 or more primer pairs are preferably used in the aboveamplification step.

[0012] Furthermore in the method for SNP typing according to the presentinvention, an Invader assay or a TaqMan PCR method is preferably used inthe above typing step.

[0013] Furthermore in the method for SNP typing according to the presentinvention, an amplification is carried out from 10 ng to 40 ng ofgenomic DNA.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Now the method for SNP typing according to the present inventionwill be further described in detail.

[0015] In the method for SNP typing according to the present invention,first, an amplification step is performed to simultaneously amplify aplurality of nucleotide sequences comprising at least one or more SNPsites using genomic DNA to be analyzed and a plurality of primer pairs.Then a typing step is performed for typing using the nucleotidesequences amplified in the amplification step.

[0016] 1. Amplification Step:

[0017] In the method of the present invention, genomic DNA to beanalyzed can be extracted by using standard, known techniques. GenomicDNA to be analyzed can also be extracted by isolating leucocytes fromperipheral blood collected from a human, and extracting according tostandard techniques from the isolated leucocytes. Particularly whenhundreds of thousands of SNP sites are genotyped, approximately severaltens of micrograms of genomic DNA is prepared in the method of theinvention. In other word, the method of the invention can perform typingof hundreds of thousands of SNPs from several ml of peripheral blood.

[0018] In the amplification step, a so-called multiplex polymerase chainreaction (multiplex PCR) is performed using genomic DNA prepared as atemplate DNA and a plurality of primer pairs for amplifying nucleotidesequences containing SNP sites to be genotyped. A preferred plurality ofprimer pairs are designed so as to be able to amplify 100 to 1500 bp DNAfragments flanking an SNP site. Each of these primers preferablycomprises 17 to 25 nucleotides, more preferably, 18 to 22 nucleotides,respectively. These primers are designed so as to flank an SNP site tobe genotyped, based on, for example, nucleotide sequence informationaccumulated in a database such as the GenBank.

[0019] The amplification step is performed by thermally denaturingtemplate DNA (genomic DNA), followed by repetition of a cycle consistingof a thermal denaturation process to denature the template DNA, anannealing process to precisely anneal a plurality of primers, and anextension process to synthesize a DNA strand from the annealed primers.Finally, another extension step is performed to further extend the DNAstrand. In addition, an appropriate temperature and time are preferablyset separately for each process.

[0020] In such an amplification step, a preferred amount of a templateDNA is 10 to 40 ng when, for example, nucleotide sequences of 100regions are amplified using 100 pairs of primers. When the amount of atemplate DNA is less than 10 ng, amplifying all 100 regions would bedifficult. In other words, with 10 ng or more of a template DNA, DNAfragments can be amplified in an amount sufficient to perform the typingstep described later. When the amount of template DNA exceeds 40 ng, itbecomes impractical to genotype hundreds of thousands of SNP sitesbecause a large amount of genomic DNA is required.

[0021] In such an amplification step, a so-called hot start method ispreferably applied. The hot start method is a technique in which theextension reaction with DNA polymerase is started when a reactionsolution reaches a temperature high enough to prevent an annealing errorand dimerization of primers. Examples of the hot start method include amethod in which at least one kind of composition essential for PCR isadded only when a reaction solution reaches a high temperature, a methodwhich uses a wax barrier, and a method which uses a monoclonal antibodyfor DNA polymerase.

[0022] In the method using a wax barrier, first in a reaction container,solid wax divides an upper layer solution containing DNA polymerase andtemplate DNA from a lower layer solution containing a primer and dNTP.Subsequently, PCR is allowed to proceed only after wax is melted byheating to a certain temperature so as to mix the upper and lower layersolutions.

[0023] In the method using a monoclonal antibody for DNA polymerase, DNApolymerase and a monoclonal antibody are bound to each other, and thusDNA polymerase is inactive until a reaction solution reaches a certaintemperature. When the reaction solution is heated to a certaintemperature (approximately 70° C. or more), the monoclonal antibody isirreversibly, thermally denatured and released from DNA polymerase.Thus, DNA polymerase is activated and PCR proceeds.

[0024] In any of these methods, the hot start method can preventextension reaction from proceeding where annealing errors occur for eachprimer, or dimerization of primers to each other. Therefore,amplification of undesired DNA fragments can be prevented.

[0025] When the above-mentioned hot start method is applied in theamplification step, nucleotide sequences of 300 or more regions can beamplified simultaneously using 300 or more primer pairs. Hence, themethod of the present invention enables typing of a greater number ofSNPs, because application of the above hot start method allowsamplification of a greater number of nucleotide sequences at one time.

[0026] 2. Typing Step:

[0027] The typing step is a process to genotype a plurality of SNPsusing DNA fragments amplified in the above-mentioned amplification step.In the method of the present invention, typing can be performed, forexample, by applying a TaqMan PCR method or an Invader assay using theDNA fragments obtained in the amplification step.

[0028] The TaqMan PCR method utilizes PCR using a fluorescence-labeledallele-specific oligonucleotide (hereinafter, referred to as TaqManprobe), a template DNA containing an SNP to be genotyped, and Taq DNApolymerase (Livak, K. J. Genet. Anal. 14, 143 (1999); Morris T. et al.,J. Clin. Microbiol. 34, 2933 (1996)). A TaqMan probe is designed basedon SNP information, and which has a 5′ end labeled with fluorescentreporter dye R, such as FAM or VIC, and a 3′ end labeled with quencher Q(FIG. 1) at the same time. Since, in this condition, the quencherabsorbs fluorescence energy, fluorescence from the TaqMan probe cannotbe detected. Further, since the 3′ end of the TaqMan probe isphosphorylated, no extension reaction occurs from the TaqMan probeduring PCR reaction (FIG. 1).

[0029] The following reactions occur when PCR is performed for atemplate DNA using the above described TaqMan probe, primers designed toamplify a region containing SNP site, and Taq DNA polymerase. First, theTaqMan probe hybridizes to a sequence specific to the template DNA (FIG.2a), while extension reaction occurs from the PCR primer hybridizing tothe template DNA (FIG. 2b). At this time, Taq DNA polymerase having 5′nuclease activity cleaves nucleotides containing fluorescent reporterdye R of the TaqMan probe when extension reaction from PCR primersreaches the 5′ end of the TaqMan probe (FIG. 2c). When a nucleotidecontaining fluorescent reporter dye R is cleaved as described above, thefluorescent reporter dye R becomes unaffected by quencher Q, and emits ameasurable fluorescence signal. Hence, detection of fluorescence of thefluorescent reporter dye R by a fluorescent detector enablesconfirmation of hybridization of the TaqMan probe and the template DNA.

[0030] For example, as shown in FIG. 3, an SNP site is supposed to bepresent at A in allele 1 (supposed to be allele 1) and that at G inallele 2 (supposed to be allele 2). While a TaqMan probe specific toallele 1 is labeled with FAM, a TaqMan probe specific to allele 2 islabeled with VIC (FIG. 3). The two types of TaqMan probe are added toPCR reagent, and then TaqMan PCR is performed for a template DNAcontaining SNP to be genotyped. PCR causes a nucleotide containingfluorescent reporter dye R to be released from a TaqMan probe having anucleotide sequence complementary to SNP site to be genotyped, so thatfluorescence is emitted. Then, fluorescent intensity of FAM and that ofVIC are measured using a fluorescent detector.

[0031] As a result, SNP in the template DNA can be genotyped as ahomozygote of allele 1 when strong fluorescence of FAM and almost nofluorescence of VIC are detected with a fluorescent detector. SNP in atemplate DNA can also be genotyped as a heterozygote of allele 1 andallele 2 when fluorescence of both FAM and VIC is detected with afluorescent detector. Further, SNP in the template DNA can be genotypedas a homozygote of allele 2 when strong fluorescence of VIC and almostno fluorescence of FAM are detected with a fluorescent detector.

[0032] On the other hand, the Invader assay uses DNA comprising the SNPto be genotyped, two types of reporter probes specific to each allele ofSNP to be genotyped, one type of Invader probe, and enzyme havingspecial endonuclease activity to cleave by recognizing DNA structure(Livak, K. J. Biomol. Eng. 14, 143-149 (1999); Morris T. et al., J.Clin. Microbiol. 34, 2933 (1996); Lyamichev, V. et al., Science, 260,778-783 (1993) and the like). In the Invader assay, SNP site can begenotyped by hybridization of an allele-specific oligonucleotide and DNAcontaining SNP to be genotyped. The Invader assay uses two types ofunlabeled oligo and one type of fluorescence-labeled oligo. One of thetwo types of unlabeled oligo is called an allele probe.

[0033] An allele probe comprises a 3′ hybridizing region whichhybridizes to a genomic DNA (template DNA) to form a complementarystrand, and a 5′ region (called FLAP) which has a sequence unrelated tothe sequence of a genomic DNA and does not hybridize to the genomic DNA.A nucleotide located at the 5′ end of the hybridizing region correspondsto an SNP (FIG. 4a). That is, an allele probe is provided with thehybridizing region which can form a complementary strand with a regionon the 5′ side from the SNP site of genomic DNA (“A” in FIG. 4a), andFlap region which has been added to the 5′ side of a nucleotide (“T” inFIG. 4a) corresponding to the SNP. Here, the term “Flap” is anoligonucleotide having a sequence complementary to a given region of aFRET probe which is described later.

[0034] Another unlabeled oligo is called an Invader probe. The Invaderprobe is designed so as to complementarily hybridize in a direction fromthe SNP site (“A” in FIG. 4b) to the 3′ end of a genomic DNA (FIG. 4b).Here, a sequence (“N” in FIG. 4b) corresponding to an SNP site may be anarbitrary nucleotide. Thus, hybridization of a genomic DNA as a templateand the above two probes causes one nucleotide (N) of the invader probeto invade into the SNP site (FIG. 4c).

[0035] A fluorescence-labeled oligo is a sequence totally independentfrom genomic DNA, and the sequence is a common sequence regardless ofthe type of SNP. This fluorescence-labeled oligo is called a FRET probe(fluorescence resonance energy transfer probe) (FIG. 5). A nucleotide(reporter, located at the 5′ terminus of FRET probe) is labeled withfluorescent dye (R), and quencher (Q) is bound upstream of thenucleotide. In this condition, no fluorescence can be detected with afluorescent detector since the quencher absorbs fluorescence.

[0036] A certain region (region 1) from the 5′ end (reporter nucleotide)of FRET probe is designed to face a region (region 2) on the 3′ side ofthe region 1 so as to form a complementary sequence. Hence, in FRETprobe, region 1 forms a complementary strand with region 2 (FIG. 5).Moreover, a region located further on the 3′ side of the region forminga complementary strand, that is, the 3′ terminal side of region 2 isdesigned so as to be able to form a complementary strand by hybridizingto Flap of an allele probe (FIG. 5).

[0037] The Invader assay uses Cleavase which is an enzyme (5′nucleotidase) having special endonuclease activity to recognize andcleave a specific structure of DNA. Cleavase can cleave the 3′ side ofan SNP position of an allele probe when a genomic DNA, allele probe andInvader probe overlap three fold at the SNP position. That is when threenucleotides overlap as shown in FIG. 4c, cleavase recognizes a portionat which the 5′ end is Flap-shaped, and cleaves the Flap portion.Therefore, cleavase recognizes this structure of an SNP site (FIG. 6a),the allele probe is cleaved at a nucleotide corresponding to the SNPsite, and then the Flap portion is released (FIG. 6b).

[0038] Next, the Flap portion released from the allele probe bindscomplementarily to the FRET probe since it has a sequence complementaryto that of the FRET probe (FIG. 6c). At this time, the SNP site of Flapinvades into the complementary binding site of FRET itself. Again,cleavase recognizes the structure and cleaves a reporter nucleotidehaving fluorescent dye. The cleaved fluorescent dye becomes unaffectedby the quencher and emits a measurable fluorescence signal (FIG. 6d). Inaddition, when a nucleotide corresponding to an SNP of allele probe doesnot match an SNP site, as shown in FIG. 7, the allele probe is notcleaved and Flap is not released since no specific DNA structure, whichis specifically recognized by cleavase, is formed. Therefore, in thiscase almost all the fluorescent dye is still bound to the reporternucleotide, and can emit any only low fluorescence signal.

[0039] Specifically, when an SNP site can be T or C, an Invader probe,an allele probe for T, and a FRET probe in which FAM has been bound to areporter nucleotide corresponding to SNP is prepared. Separately, anInvader probe, an allele probe for C, and a FRET probe in which VIC hasbeen bound to a reporter corresponding to SNP is prepared. All of themare mixed and the Invader assay is performed. An SNP site is thengenotyped by detecting fluorescent intensity of FAM and that of VIC witha fluorescence detector. That is when strong fluorescence of FAM isdetected but very low fluorescence of VIC is detected, the SNP can begenotyped as a homozygote (T/T). When fluorescence of both FAM and VICcan be detected, the SNP can be genotyped as a heterozygote (T/C).Further, when strong fluorescence of VIC is detected but very lowfluorescence of FAM is detected, the SNP can be genotyped as ahomozygote (T/T).

[0040] Now, a so-called SniPer method may be used in the typing step.The SniPer method is based on a technique called RCA (rolling circleamplification) method. In the SniPer method, DNA polymerase sequentiallysynthesizes a complementary strand DNA while migrating on a circularsingle stranded DNA as a template. According to the method, SNP can begenotyped by measuring the presence or absence of coloring reaction dueto DNA amplification (Lizardi, P. M. et al., Nature Genet., 19, 225-232(1998); Piated, A. S. et al., Nature Biotech., 16, 359-363 (1998)).

[0041] Particularly in the typing step, card 1, as shown in FIG. 8a, ispreferably used when any of the methods described above is employed.Card 1 comprises numerous wells 2, arranged in a matrix on a primarysurface thereof, wall surfaces 3 which are so formed as to projectaround each well 2, and a plurality of grooves 4 which are formedbetween adjacent wells 2. An example of a card 1 that can be used is,but is not limited to, a card 1 comprising a total of 384 wells 2 (24columns×16 lines) formed thereon.

[0042] When the typing step is performed using card 1, first the DNAfragment obtained in the above-mentioned amplification step is dispensedinto wells 2, in the form of PCR reaction solution. Then, the dispensedPCR reaction solution is dried up, so that the dried DNA fragmentsremain on the bottom surfaces of wells 2.

[0043] Subsequently, as shown in FIG. 8b, a reagent necessary for thetyping step, for example, a reagent necessary for the Invader assay isdispensed into wells 2. At this time, the reagent necessary for thetyping step is dispensed onto the upper portion of a wall surface 3 in avolume exceeding that of well 2 but not such that it overflows, due tosurface tension. Further, the DNA fragment present in a dried state inwell 2 is dissolved in the reagent dispensed into wells 2, necessary forthe typing step. When a reagent necessary for the typing step isdispensed into wells 2, a pipette preferably used herein is anon-contact dispensing equipment which is capable of simultaneouslydispensing a solution into a plurality of wells 2 arranged on card 1.Since a non-contact dispensing equipment does not contact with theinside of wells 2, such a pipette can prevent contamination fromoccurring between a plurality of cards 1.

[0044] Next as shown in FIG. 8c, plastic plate 5 which is large enoughto cover the primary surface of card 1 is overlayed on the primarysurface of card 1. Accordingly, the reagent dispensed into wells 2 flowsout toward the outside of wells 2. Most of the reagent that has flowedout remains within grooves 4.

[0045] Then, card 1 with plastic plate 5 overlayed on the primarysurface thereof is ultrasonically treated with a ultrasonic weldingequipment, so that plastic plate 5 and card 1 are welded. Asspecifically shown in FIG. 8d, the upper surfaces of the wall surfaces 3and the plastic plate 5 are in contact with each other, and welding canbe performed at the contacting area.

[0046] As described above, the use of card 1 in the typing step enablesprevention of foaming within wells 2 and prevents the reagent dispensedwithin wells 2 from flowing out even when card 1 is subjected to heattreatment. Therefore, card 1 employed in the typing step can improvedetection sensitivity for a fluorescent dye or the like.

[0047] Further, use of card 1 provided with well 2 whose volume is 0.6ml can largely decrease the volume of reagent necessary for the typingstep, so as to largely reduce the cost required for SNP typing.

[0048] Furthermore, when the typing step is performed using card 1, heattreatment or the like included in the typing step is preferablyperformed using a thermostat water bath. By using a thermostat waterbath, the typing step can be performed using numerous cards 1simultaneously. For example, since performing the typing stepsimultaneously using numerous cards 1 is difficult when a thermal cycleris used, a thermostat water bath is preferably used.

[0049] 3. Effect of the Method of the Present Invention

[0050] According to the method of the present invention, a target SNPcan be genotyped by amplifying in the amplification step genomic DNAcollected or extracted, and using the amplified DNA fragments.Therefore, the method enables typing of several hundreds of thousands ofSNP sites even with a small amount of genomic DNA. For example, when100,000 SNP sites are genotyped, approximately 10 μg of genomic DNAwould be required according to an estimation that approximately 0.1 ngof genomic DNA is required per SNP site. Approximately 10 μg of genomicDNA can be extracted from 1.25 ml of peripheral blood collected from ahuman.

[0051] On the other hand, since SNP typing using genomic DNA it self bythe Invader assay or the like requires several tens ng of genomic DNAper SNP site, several mg of genomic DNA must be prepared to genotype100,000 SNP sites. To obtain several mg of genomic DNA, 500 ml or moreperipheral blood must be collected. However, it is actually impossibleto prepare genomic DNA in such a volume.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 schematically shows TaqMan probes.

[0053]FIG. 2 shows the outline of each steps composing the TaqMan PCRmethod.

[0054]FIG. 3 schematically shows fluorescence-labeled TaqMan probes.

[0055]FIG. 4 schematically shows the Invader assay.

[0056]FIG. 5 schematically shows a FRET probe.

[0057]FIG. 6 schematically shows the Invader assay.

[0058]FIG. 7 schematically shows a probe which does not match an allele.

[0059]FIG. 8 shows a partial cross-sectional view of a card used in thetyping step.

[0060]FIG. 9 is a characteristic figure showing the result of typing SNPNo. 1.

[0061]FIG. 10 is a characteristic figure showing the result of typing bythe Invader assay directly using genomic DNA.

[0062]FIG. 11 is a flowchart of the method for typing described inexample 4.

[0063]FIG. 12 is a characteristic figure showing the result of detectinga signal intensity of VIC/ROX and FAM/ROX in example 4.

EXAMPLE

[0064] Now the present invention will be described more specifically,but the technical scope of the invention is not limited by the followingexamples.

Example 1 Preparation of Genomic DNA

[0065] Leukocytes were isolated from the peripheral blood collected froma subject who had given informed consent, and genomic DNA was extractedtherefrom. Genomic DNA was extracted according to Laboratory Manual forGenomic Analysis (Yusuke Nakamura ed., Springer-Verlag Tokyo) asdescribed below. 10 ml of the blood was transferred to a 50 ml Falcontube, and then centrifuged at 3,000 rpm for 5 min at room temperature.The supernatant (serum) was discarded with a pipette, 30 ml of RBC lysisbuffer (10 mM NH₄HCO₃, 144 mM NH₄Cl) was added thereto. After mixing todisperse the precipitate well, the mixture was allowed to stand at roomtemperature for 20 min. Subsequently, centrifugation was performed at3,000 rpm for 5 min at room temperature, the supernatant (serum) wasdiscarded with a pipette, thereby obtaining a leukocyte pellet. 30 ml ofRBC lysis buffer was added to the pellet, and then a similar procedurewas performed twice. 4 ml of Proteinase K buffer (50 mM Tris-HCl(pH7.4), 100 mM NaCl, 1 mM EDTA(pH 8.0)), 200 μl of 10% SDS, 200 μl of 10mg/ml Proteinase K were added to the leukocyte pellet. The mixture wasmixed by inverting, and allowed to stand at 37° C. overnight. 4 ml ofphenol was added to the mixture, and then mixed by slowly inverting for4 hours with a rotator (Rotator T-50, Taitec). Centrifugation wasperformed at 3,000 rpm for 10 min at room temperature, and the upperlayer was collected in a new tube. 4 ml of phenol-chloroform-isoamylalcohol (volume ratio 25:24:1) was added to the product, followed by twohours of similar mixing by inverting and centrifugation. The upper layerwas collected in a new tube, and 4 ml of chloroform-isoamyl alcohol(volume ratio 24:1) was added thereto. Then the product was mixed byinverting similarly for 30 min, followed by centrifugation. The upperlayer was collected in a new tube, and 400 μl of 8 M ammonium acetateand 4 ml of isopropanol were added thereto, followed by mixing byinverting. White, filamentous precipitate (DNA) was collected in a 2 mltube, 1 ml of 70% ethanol was added thereto, followed by mixing byinverting. DNA was collected in a new 2 ml tube and then air-dried. DNAwas dissolved in 500 μl of TE solution (10 mM Tris-HCl (pH 7.4), 1 mMEDTA (pH7.4)), thereby preparing a genomic DNA sample.

Example 2 Amplification of Genomic DNA

[0066] PCR was performed with a 50 μl system using 40 ng of the genomicDNA obtained in Example 1. A reaction solution contains 200 types ofprimer (50 pmol 100 pairs, SEQ ID NOS: 1 to 200), 10 units of EX-TaqDNApolymerase (Takara Shuzo), and 0.55 μg of TaqStart (CLONTECHLaboratories). TaqStart is an antibody for EX-TaqDNA polymerase. The hotstart method can be performed by adding TaqStart to the reactionsolution.

[0067] PCR was performed with GeneAmp PCR system 9700 (AppliedBiosystems). DNA was denatured at 94° C. for 2 min, a cycle consistingof a denaturation process at 94° C. for 15 sec, an annealing process at60° C. at 45 sec, and then an extension process at 72° C. for 3 min wasrepeated 35 times, followed by extension at 72° C. for 3 min.

[0068] As shown in Table 1, a plurality of DNA fragments containing SNPIdentification Nos. (“SNP ID” in Table 1) 1 to 100 can be amplifiedsimultaneously. TABLE 1 SNP ID Forward primer Reverse primer SNP name 1SEQ ID NO 1 SEQ ID NO 2 AC000353.27_20000214_5_24737 2 SEQ ID NO 3 SEQID NO 4 AC000388.1_19970529_9_37703 3 SEQ ID NO 5 SEQ ID NO 6AC001643.1_19970529_3_6293 4 SEQ ID NO 7 SEQ ID NO 8AC002237.1_19970606_1_1204 5 SEQ ID NO 9 SEQ ID NO 10AC002319.1_19980203_3_29222 6 SEQ ID NO 11 SEQ ID NO 12AC002364.1_19981204_2_117944 7 SEQ ID NO 13 SEQ ID NO 14AC003005.1_19971022_1_2731 8 SEQ ID NO 15 SEQ ID NO 16AC003005.1_19971022_3_5667 9 SEQ ID NO 17 SEQ ID NO 18AC003689.1_19981121_2_45471 10 SEQ ID NO 19 SEQ ID NO 20AF066064.1_19980603_1_563 11 SEQ ID NO 21 SEQ ID NO 22AF077374.1_19990202_1_1708 12 SEQ ID NO 23 SEQ ID NO 24AF157101.1_19990624_1_618 13 SEQ ID NO 25 SEQ ID NO 26AF196968.1_19991109_1_6368 14 SEQ ID NO 27 SEQ ID NO 28AJ009610.1_19990104_4_26810 15 SEQ ID NO 29 SEQ ID NO 30AJ011772.1_19981005_2_903 16 SEQ ID NO 31 SEQ ID NO 32AJ011931.1_19981110_5_23638 17 SEQ ID NO 33 SEQ ID NO 34AJ229043.1_19990122_1_3475 18 SEQ ID NO 35 SEQ ID NO 36AL008633.1_19971029_1_33923 19 SEQ ID NO 37 SEQ ID NO 38AL008634.1_19981109_13_92880 20 SEQ ID NO 39 SEQ ID NO 40AL008634.1_19981109_13_93343 21 SEQ ID NO 41 SEQ ID NO 42AL008634.1_19981109_14_95554 22 SEQ ID NO 43 SEQ ID NO 44AL008638.1_19981123_4_52385 23 SEQ ID NO 45 SEQ ID NO 46AL008730.1_19980204_2_66080 24 SEQ ID NO 47 SEQ ID NO 48AL008733.10_19991225_1_5608 25 SEQ ID NO 49 SEQ ID NO 50AL008734.10_19990610_1_7867 26 SEQ ID NO 51 SEQ ID NO 52AL021917.1_19980721_19_77217 27 SEQ ID NO 53 SEQ ID NO 54AL021937.1_19990303_93_124364 28 SEQ ID NO 55 SEQ ID NO 56AL022721.1_19990324_13_73842 29 SEQ ID NO 57 SEQ ID NO 58AL023279.1_19990305_3_69539 30 SEQ ID NO 59 SEQ ID NO 60AL049557.19_73359 31 SEQ ID NO 61 SEQ ID NO 62 AL049569.13_164971 32 SEQID NO 63 SEQ ID NO 64 AL049569.13_61322 33 SEQ ID NO 65 SEQ ID NO 66AL049569.13_61680 34 SEQ ID NO 67 SEQ ID NO 68 AL049569.13_61971 35 SEQID NO 69 SEQ ID NO 70 AL049569.13_62026 36 SEQ ID NO 71 SEQ ID NO 72AL049569.13_87106 37 SEQ ID NO 73 SEQ ID NO 74 AL049569.13_87279 38 SEQID NO 75 SEQ ID NO 76 AL049569.13_88461 39 SEQ ID NO 77 SEQ ID NO 78AL049569.13_88502 40 SEQ ID NO 79 SEQ ID NO 80 AL049575.7_10509 41 SEQID NO 81 SEQ ID NO 82 AL049611.24_75054 42 SEQ ID NO 83 SEQ ID NO 84AL049611.24_75895 43 SEQ ID NO 85 SEQ ID NO 86 AL049612.11_45784 44 SEQID NO 87 SEQ ID NO 88 AL049649.4_93434 45 SEQ ID NO 89 SEQ ID NO 90AL049649.4_93918 46 SEQ ID NO 91 SEQ ID NO 92 AL049650.8_62150 47 SEQ IDNO 93 SEQ ID NO 94 AL049691.17_64637 48 SEQ ID NO 95 SEQ ID NO 96AL049694.9_4336 49 SEQ ID NO 97 SEQ ID NO 98 AL049698.3_3216 50 SEQ IDNO 99 SEQ ID NO 100 AL049698.3_3822 51 SEQ ID NO 101 SEQ ID NO 102AL049758.11_67143 52 SEQ ID NO 103 SEQ ID NO 104 AL049758.11_79044 53SEQ ID NO 105 SEQ ID NO 106 AL049759.10_111608 54 SEQ ID NO 107 SEQ IDNO 108 AL049795.20_113584 55 SEQ ID NO 109 SEQ ID NO 110AL049829.2_138544 56 SEQ ID NO 111 SEQ ID NO 112 AL049829.2_161140 57SEQ ID NO 113 SEQ ID NO 114 AL049843.18_49141 58 SEQ ID NO 115 SEQ ID NO116 AL096766.12_13162 59 SEQ ID NO 117 SEQ ID NO 118 AP000065.1_58129 60SEQ ID NO 119 SEQ ID NO 120 AP000168.1_56285 61 SEQ ID NO 121 SEQ ID NO122 AP000171.1_87106 62 SEQ ID NO 123 SEQ ID NO 124 AP000347.1_81990 63SEQ ID NO 125 SEQ ID NO 126 AP000349.1_19017 64 SEQ ID NO 127 SEQ ID NO128 AP000350.1_10554 65 SEQ ID NO 129 SEQ ID NO 130 AP000350.1_10756 66SEQ ID NO 131 SEQ ID NO 132 AP000350.1_11294 67 SEQ ID NO 133 SEQ ID NO134 AP000350.1_31581 68 SEQ ID NO 135 SEQ ID NO 136 AP000352.1_63635 69SEQ ID NO 137 SEQ ID NO 138 AP000353.1_86203 70 SEQ ID NO 139 SEQ ID NO140 AP000355.1_132012 71 SEQ ID NO 141 SEQ ID NO 142 AP000493.1_12911472 SEQ ID NO 143 SEQ ID NO 144 AP000495.1_60416 73 SEQ ID NO 145 SEQ IDNO 146 AP000500.1_113211 74 SEQ ID NO 147 SEQ ID NO 148AP000500.1_113401 75 SEQ ID NO 149 SEQ ID NO 150 AP000500.1_194483 76SEQ ID NO 151 SEQ ID NO 152 AP000500.1_25277 77 SEQ ID NO 153 SEQ ID NO154 AP000501.1_37357 78 SEQ ID NO 155 SEQ ID NO 156 AP000501.1_99530 79SEQ ID NO 157 SEQ ID NO 158 AP001041.1_6501 80 SEQ ID NO 159 SEQ ID NO160 AP001041.1_6582 81 SEQ ID NO 161 SEQ ID NO 162 AP001054.1_35804 82SEQ ID NO 163 SEQ ID NO 164 AP001054.1_36083 83 SEQ ID NO 165 SEQ ID NO166 AP001054.1_36142 84 SEQ ID NO 167 SEQ ID NO 168 AP001101.1_12400 85SEQ ID NO 169 SEQ ID NO 170 D42052.1_7718 86 SEQ ID NO 171 SEQ ID NO 172D50561.1_1218 87 SEQ ID NO 173 SEQ ID NO 174 D50561.1_564 88 SEQ ID NO175 SEQ ID NO 176 NT_002717.1_29435 89 SEQ ID NO 177 SEQ ID NO 178U07563.1_68521 90 SEQ ID NO 179 SEQ ID NO 180 X56832.1_2826 91 SEQ ID NO181 SEQ ID NO 182 X69299.1_1633 92 SEQ ID NO 183 SEQ ID NO 184X74107.1_29168 93 SEQ ID NO 185 SEQ ID NO 186 X74107.1_29545 94 SEQ IDNO 187 SEQ ID NO 188 X78901.1_1934 95 SEQ ID NO 189 SEQ ID NO 190X87344.1_115764 96 SEQ ID NO 191 SEQ ID NO 192 X91863.1_2477 97 SEQ IDNO 193 SEQ ID NO 194 Y08378.1_1560 98 SEQ ID NO 195 SEQ ID NO 196Y12852.1_4439 99 SEQ ID NO 197 SEQ ID NO 198 Y16792.1_4230 100 SEQ ID NO199 SEQ ID NO 200 Z54246.1_8005

Example 3 Typing by Invader Assay

[0069] The sample obtained by PCR as described in Example 2 wasapportioned, 0.2 μl each, to 100 tubes and then typing was performed for100 types of SNPs using an Invader assay kit (Third Wave Technology).That is, 0.5 μl of the sample was added to the kit containing 0.5 μl ofsignal buffer, 0.5 μl of FRET probe, 0.5 μl of structure-specificdeoxyribonuclease, and 1 μl of allele-specific probe. The reactionvolume was prepared to be 10 μl. FRET probes were labeled with differentfluorescent dyes (FAM and VIC). Two types of FRET probes differing intheir Flap complementary sequences were used. A pair of probes has Flapportions corresponding to two types of FRET probes. Next, the reactionsolution was incubated at 95° C. for 5 min, and then 63° C. for 15 minusing ABI7700 (Applied Biosystems). Fluorescence emitted duringincubation was detected using the device.

[0070]FIGS. 9a, b and c show respectively the results of typing threedifferent samples using SNP ID NO: 1 probe. In FIG. 9, a continuous linedenotes fluorescence of FAM, and a broken line denotes that of VIC. Asshown in FIG. 9a, only the fluorescence of VIC was elevated for thissample. This result suggested that both alleles of nucleotides (SNP IDNO: 1) in this sample corresponded to a specific probe having a Flapcomplementary to FRET probe labeled with VIC, that is, the sample washomozygous. The result for the sample in FIG. 9b suggested that one ofthe alleles corresponded to a specific probe having a Flap complementaryto FAM-labeled FRET probe, and the other allele corresponded to aspecific probe having a Flap complementary to VIC-labeled FRET probe,that is, this sample was heterozygous. Further, the sample in FIG. 9cwas shown to be homozygous corresponding to a specific probe having aFlap complementary to FAM-labeled FRET probe.

[0071] Moreover, fluorescence was detected for 98% of SNPs (SNP ID NOS:1 to 100). The result suggested that with a very small amount of genomicDNA, 0.4 ng per SNP, typing was possible by the method of the presentinvention.

[0072] When the Invader assay was directly performed using 0.4 ng ofgenomic DNA, no fluorescence was detected and typing was impossible asshown in FIG. 10. Probably, this was due to the amount of DNA (0.4 ng)used for assay being insufficient to obtain fluorescence required fordetection.

Example 4 Improved Method

[0073] A typing system using a smaller quantity of genomic DNA wasstudied by improving the typing system described in Example 3. In thisexample, a 96-well PCR plate was used and 96 DNA fragments wereamplified in each well with a single amplification reaction. Inaddition, the flow chart of the typing system performed in this Example4 is shown in FIG. 11.

[0074] The PCR product obtained in Example 2 was diluted and thentransferred into a 384 deep well. Then 0.8 μl of the PCR product wasdispensed into each well (volume: 0.6 μl) of a card shown in FIG. 8a. Anautomatic liquid handling system, Tango (Robbins), was used fordispensing. The PCR product was dispensed using an automatic liquidhandling system Tango from a single plate having 384 deep wells to 96cards. The automatic liquid handling system Tango is capable ofsimultaneously dispensing 384 samples, that is, capable of dispensinginto 8 cards in a single operation. Thereafter, the dispensed PCRproduct was naturally dried at room temperature.

[0075] Next, 0.03 μl of signal buffer, 0.03 μl of FRET probe, 0.03 μl ofstructure specific deoxyribonuclease and 0.06 μl of allele specificprobe contained in an Invader assay kit (Third Wave Technology) weredispensed into wells. These solutions were dispensed using a non-contactdispensing workstation PixSys4200 (Cartesian Technologies). Thenon-contact dispensing workstation PixSys4200 is capable ofsimultaneously dispensing the above solutions into 8 cards, each having384 wells.

[0076] Subsequently, a plastic plate was overlayed on the card, and thenultrasonic welding using ultrasonic welding equipment (Branson) wasperformed. Thus, 96 cards, each having 384 wells, could be prepared.Then, incubation was performed using a thermobath (TAITEC) at 95° C. for5 min, followed by 63° C. for 60 min. After incubation, fluorescenceemitted from each well of the cards was detected using a fluorescencedetector ABI 7900(Applied Biosystems), and typing was performed. FIG. 12shows the result detecting a signal intensity of VIC/ROX and FAM/ROX. InFIG. 12, the horizontal axis shows the signal intensity of VIC/ROX; thevertical axis shows the signal intensity of FAM/ROX. The bottom rightcluster of spots indicates a strong VIC/ROX signal and a weak FAM/ROXsignal; the samples in this cluster are judged to be homozygous forallele 1. Similarly, samples indicated by the top right cluster of spotsare heterozygous, and samples indicated by the top left cluster of spotsare homozygous for allele 2.

[0077] Although only 0.1 ng genomic DNA was used as template, allelescould be discriminated clearly.

[0078] According to Example 4, as shown in FIG. 12, alleles could bediscriminated clearly and fluorescence could be detected with highsensitivity even when 0.1 ng of the genomic DNA was subjected to asingle typing, thereby allowing accurate typing. Thus, genomic DNA to besubjected to a single PCR reaction in an amplification step would beapproximately 10 ng. Therefore according to Example 4, with genomic DNAin a volume approximately ¼ of that used in the method of Example 3,typing can be performed.

[0079] Effect of the Invention

[0080] As described above in detail, the method for SNP typing accordingto the present invention can type several hundreds of thousands of SNPsites using a very small amount of genomic DNA. Hence, the method forSNP typing according to the present invention enables typing of severalhundreds of thousands of SNP sites using very small amount of genomicDNA at low cost and in a short period of time.

[0081] Sequence Listing Free Text

[0082] SEQ ID NOS: 1 to 200 are synthetic primers.

1 200 1 20 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 1 1 ccagcaggac ttggtgacag 20 2 21 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 1 2 gcaagaagca gccagatcaa g 21 3 20 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 2 3cagccaccca ctcagtcttg 20 4 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 2 4 aggtcctggc tctgcgtaac20 5 22 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 3 5 gcttgagact caccctctga tg 22 6 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 3 6 gtcccgactt gaaggtccac 20 7 22 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 4 7tctgccaagc agaaacctag ag 22 8 19 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 4 8 ggcaccttga gaggaatgc19 9 19 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 5 9 ctttccgaca acgagagcg 19 10 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 5 10 cacctggact ctgcatcctg 20 11 19 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 6 11aggccgtgag ggaatgatg 19 12 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 6 12 gggtgtctag catggtgctg20 13 21 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 7 13 ttcagcatag ctccagaagg c 21 14 22 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 7 14 tttggaccct tgtcctaaca ac 22 15 23 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 815 tggctcacta aatgcactac cac 23 16 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 8 16acctggaggt gaagcgagac 20 17 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 9 17 accacaggct ccaggaagtg20 18 19 DNA Artificial Sequence Description of Artificial SequenceReverse Primer for SNP ID 9 18 tgcgtttgca ctggtaggc 19 19 20 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 10 19 cactcccacc accatcactg 20 20 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 10 20gctcacggaa ctcgaagacg 20 21 22 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 11 21 caggtgacatcactgtcaga gc 22 22 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 11 22 acctgctgct gttgaagctg 20 23 23DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 12 23 cgttggaagc ctgtactcct tag 23 24 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 12 24 aggagagctc acccgaagtg 20 25 20 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 13 25ggcacctctc caggattgtg 20 26 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 13 26 gaagccagggcaagtcattg 20 27 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 14 27 cccaaggccc actgtgttac 20 28 20DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 14 28 cctggtgcca agtggtcaag 20 29 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 1529 gagcattgcc ctcctcactg 20 30 22 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 15 30 gtgccacaattgatatgacc ag 22 31 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 16 31 gcctctacct ttaccgtccg 20 32 20DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 16 32 gccacctccc tgtcttcatc 20 33 21 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 1733 cagcttcagg ccaaatgtat g 21 34 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 17 34 tcacacctcctcctccattg 20 35 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 18 35 tccaccctga tcaagtccag 20 36 19DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 18 36 gcatgggtgc actgttgac 19 37 22 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 1937 aaattaaggc acaggcagtg ag 22 38 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 19 38 gtcctctgctttgctcaggc 20 39 22 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 20 39 aaattaaggc acaggcagtg ag 22 4020 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 20 40 gtcctctgct ttgctcaggc 20 41 24 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 2141 tctatgtggg taggatctcc agac 24 42 21 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 21 42tcgaaacaga agatgtggct g 21 43 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 22 43 cagcagcaacaacaaccgtc 20 44 24 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 22 44 cccaagtgtg gtaggtttac aatg 2445 20 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 23 45 gaaatgcctc cctggaacag 20 46 19 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 2346 ctctgccaag cccatcttg 19 47 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 24 47 tccctgagcccaggtaagtc 20 48 21 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 24 48 tgttccctga tcctcatcca g 21 4924 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 25 49 catcctcgtc actgactaat agcg 24 50 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 25 50 tcaacagcga actccacctg 20 51 20 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 26 51gccagggact gaagctgaac 20 52 21 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 26 52 aaagcatcagtgggcagaat c 21 53 24 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 27 53 tggtgagtgg tgaggtatta gcag 2454 22 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 27 54 cactacatgg cacctcagga ag 22 55 21 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 2855 agggattcag tcagttccga g 21 56 24 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 28 56 cgttacttccaaatgtcagg agtg 24 57 24 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 29 57 cactttgagcactctcagga gaac 24 58 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 29 58 gctttgagcaaggcttccag 20 59 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 30 59 gagaacgggc tgaggacaag 20 60 20DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 30 60 tgccagagaa agggtgactg 20 61 24 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 3161 ccctgagtct agctcaaatc tctc 24 62 21 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 31 62cctgctcctt gagcttgtca c 21 63 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 32 63 ctgagggtcccttcaccaag 20 64 22 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 32 64 gcaacagcct gaatgtacac ag 22 6520 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 33 65 ctgagggtcc cttcaccaag 20 66 22 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 3366 gcaacagcct gaatgtacac ag 22 67 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 34 67 ctgagggtcccttcaccaag 20 68 22 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 34 68 gcaacagcct gaatgtacac ag 22 6920 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 35 69 ctgagggtcc cttcaccaag 20 70 22 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 3570 gcaacagcct gaatgtacac ag 22 71 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 36 71 ccactgtcctggctcagatg 20 72 21 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 36 72 gaggatgtca cggttccagt c 21 7324 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 37 73 gtgaccttcc tctgtcctat tacg 24 74 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 37 74 tttcagcagg gacagagtcg 20 75 24 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 38 75gtgaccttcc tctgtcctat tacg 24 76 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 38 76 tttcagcagggacagagtcg 20 77 24 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 39 77 gtgaccttcc tctgtcctat tacg 2478 20 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 39 78 tttcagcagg gacagagtcg 20 79 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 4079 atccactggc cattctgctg 20 80 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 40 80 gctcaaggcagactggtgtc 20 81 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 41 81 aggcagacaa atcgccactc 20 82 20DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 41 82 tgcatgggct tcagtagagc 20 83 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 4283 aggcagacaa atcgccactc 20 84 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 42 84 tgcatgggcttcagtagagc 20 85 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 43 85 tgtgggctgc tctgaggtag 20 86 20DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 43 86 cccaccctcc tttggtaatg 20 87 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 4487 gggaagaccc agccataatc 20 88 21 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 44 88 gagttggtgggcactaaggt g 21 89 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 45 89 gggaagaccc agccataatc 20 90 21DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 45 90 gagttggtgg gcactaaggt g 21 91 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 4691 ctgggcctgt gtcttcactg 20 92 21 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 46 92 ggcaaaggtcttggtgtcaa c 21 93 24 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 47 93 gcagccctct gactatatga gttg 2494 20 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 47 94 agaacgcagc aaggaagcac 20 95 23 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 4895 gattagcgtt tctttcagcc atc 23 96 22 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 48 96tctgaattcc cattcttcat gc 22 97 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 49 97 ggccaaaggttccaggagag 20 98 21 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 49 98 cgatgcagag actgtccaga g 21 9920 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 50 99 ggccaaaggt tccaggagag 20 100 21 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 50100 cgatgcagag actgtccaga g 21 101 20 DNA Artificial SequenceDescription of Artificial Sequence Forward Primer for SNP ID 51 101cctcctcagt ttctccagcg 20 102 19 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 51 102 tgggcatctgaatggaagc 19 103 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 52 103 accaatccaa gggctaggtg 20 10422 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 52 104 caggtccagc agtgatccat ac 22 105 23 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 53 105 gttacaaacc tgacttgtgg ctc 23 106 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 53106 ggctatgagt tcccgctcag 20 107 22 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 54 107 gccaaacaatccctcatgat ac 22 108 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 54 108 atgcttcctctaccatggcg 20 109 22 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 55 109 tcccaactca tttcagcatc tc 22110 20 DNA Artificial Sequence Description of Artificial SequenceReverse Primer for SNP ID 55 110 tgtctgcctc cctgactctg 20 111 20 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 56 111 ttaactggcc ctgtctggtg 20 112 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 56112 gtgcacacag aggtgtagcg 20 113 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 57 113 gggcttcttctgcatgtgtg 20 114 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 57 114 tgcttcccac tgttctcagc 20 11522 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 58 115 aaacctcact gtctgcttcc tg 22 116 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 58 116 caggtgagat cggcacactc 20 117 23 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 59117 ccacctgtaa gaacagaagt ggc 23 118 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 59 118acccaagttt gggactctgc 20 119 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 60 119 tttggccttgtttgcctctg 20 120 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 60 120 aaggccacag tttgagaacg 20 12122 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 61 121 gagtgtggtc cataaacttg gc 22 122 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 61 122 accacgtctc tagccagtcg 20 123 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 62123 gctgtgtgac gttaggccag 20 124 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 62 124 agatactgggttccatccgc 20 125 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 63 125 gttctcggag gtggctcttg 20 12622 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 63 126 ccacatcact ctctcctgca tc 22 127 20 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 64 127 gcttattcct gcaaggcgtc 20 128 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 64128 aatggaagcc aaaggcacag 20 129 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 65 129 gcttattcctgcaaggcgtc 20 130 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 65 130 aatggaagcc aaaggcacag 20 13120 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 66 131 gcttattcct gcaaggcgtc 20 132 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 66132 aatggaagcc aaaggcacag 20 133 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 67 133 aaccctgagcctgtcacctg 20 134 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 67 134 tgagccctga atgcgagtag 20 13520 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 68 135 tgtgaccttc ctggctcttc 20 136 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 68136 agcctcactg acatgccttg 20 137 21 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 69 137 caactgtgagtgaccgtgga g 21 138 23 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 69 138 agtgaggtat tggaatctga ggc 23139 21 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 70 139 caatattagc tccaccgagg c 21 140 21 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 70 140 cctcgccaac taaatgcaga c 21 141 22 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 71141 cataagccga gtggtacaga gc 22 142 23 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 71 142tccaaaggcc atagtttacc aag 23 143 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 72 143 tggcttgaggttctggcttc 20 144 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 72 144 tgtgacgggt aaggcagatg 20 14521 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 73 145 cctatgctca gccaaggtca g 21 146 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 73 146 agaaccacct gggctgctac 20 147 21 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 74147 cctatgctca gccaaggtca g 21 148 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 74 148agaaccacct gggctgctac 20 149 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 75 149 ctgtgatgggctgcagaatg 20 150 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 75 150 ggagagcctc cagttcaagc 20 15121 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 76 151 cacccagtgc agccttatag c 21 152 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 76 152 acctccctct ctgccttctg 20 153 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 77153 accacggagt ctggcatcac 20 154 24 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 77 154 cggtcagaacaaagagagtg gaac 24 155 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 78 155 tttgtccttgggcttggtag 20 156 22 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 78 156 cagggagagg tatacgatgg tg 22157 22 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 79 157 cccatcccgt taaagcactt ag 22 158 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 79 158 aggatgggct tcccactcag 20 159 22 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 80159 cccatcccgt taaagcactt ag 22 160 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 80 160aggatgggct tcccactcag 20 161 24 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 81 161 gtgtgctttgtttggtttgc atag 24 162 19 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 81 162 ctgggaatgtgccagcaag 19 163 24 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 82 163 gtgtgctttg tttggtttgc atag 24164 19 DNA Artificial Sequence Description of Artificial SequenceReverse Primer for SNP ID 82 164 ctgggaatgt gccagcaag 19 165 24 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 83 165 gtgtgctttg tttggtttgc atag 24 166 19 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID 83166 ctgggaatgt gccagcaag 19 167 20 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 84 167 ccgtgggaacatcctctgtg 20 168 20 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 84 168 gggtttgcag aatcagcctc 20 16921 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 85 169 ggcacacttg agcacttgat g 21 170 21 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 85 170 ggaggacaca cagaggaatg c 21 171 23 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 86171 caaacaggtc acatttgctg aag 23 172 22 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 86 172tggcccacac agactaataa gc 22 173 23 DNA Artificial Sequence Descriptionof Artificial Sequence Forward Primer for SNP ID 87 173 caaacaggtcacatttgctg aag 23 174 22 DNA Artificial Sequence Description ofArtificial Sequence Reverse Primer for SNP ID 87 174 tggcccacacagactaataa gc 22 175 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 88 175 ggaggcctgacagccatatc 20 176 21 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 88 176 gccatatgtg gaacaagcag c 21 17721 DNA Artificial Sequence Description of Artificial Sequence ForwardPrimer for SNP ID 89 177 ttctttctgc catcaagttg c 21 178 20 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 89 178 gctttgccag gagcctagtg 20 179 22 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 90179 tgtgatcttc caattcctcc tg 22 180 20 DNA Artificial SequenceDescription of Artificial Sequence Reverse Primer for SNP ID 90 180tatggcaggg aaggaagcac 20 181 20 DNA Artificial Sequence Description ofArtificial Sequence Forward Primer for SNP ID 91 181 tggtgaagctgctggatgac 20 182 24 DNA Artificial Sequence Description of ArtificialSequence Reverse Primer for SNP ID 91 182 gacacccacc aaagcatgta taac 24183 20 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 92 183 tgcaccagac agggtagctg 20 184 346 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 92 184 ccatccagcc aagtccttgt agtgcaccag acagggtagc tgccatccagccaagtcctt 60 gtagccagac ggcttagagc actgcgagag catccaccag agtgggacggatgaagatga 120 acgcatccca gcagccctct tagcttgcct tgaacttgct ctgccacacctgccctttat 180 tggtctctcc agcaggtaca ggcacgcctt catctctgca agctccctcatgtcctggtg 240 cattgagctg cgaagagagc cagaggatca caggtcgtag gcagatgccatccagactgg 300 gtcagtgctc catgtgggaa tcggtgtcaa ggcacatcac atggtc 346185 20 DNA Artificial Sequence Description of Artificial SequenceForward Primer for SNP ID 93 185 tgcaccagac agggtagctg 20 186 22 DNAArtificial Sequence Description of Artificial Sequence Reverse Primerfor SNP ID 93 186 ccatccagcc aagtccttgt ag 22 187 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 94187 ccagacggct tagagcactg 20 188 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 94 188 cgagagcatccaccagagtg 20 189 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 95 189 ggacggatga agatgaacgc 20 19020 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 95 190 atcccagcag ccctcttagc 20 191 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 96191 ttgccttgaa cttgctctgc 20 192 21 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 96 192 cacacctgccctttattggt c 21 193 20 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 97 193 tctccagcag gtacaggcac 20 19420 DNA Artificial Sequence Description of Artificial Sequence ReversePrimer for SNP ID 97 194 gccttcatct ctgcaagctc 20 195 20 DNA ArtificialSequence Description of Artificial Sequence Forward Primer for SNP ID 98195 cctcatgtcc tggtgcattg 20 196 20 DNA Artificial Sequence Descriptionof Artificial Sequence Reverse Primer for SNP ID 98 196 agctgcgaagagagccagag 20 197 22 DNA Artificial Sequence Description of ArtificialSequence Forward Primer for SNP ID 99 197 gatcacaggt cgtaggcaga tg 22198 20 DNA Artificial Sequence Description of Artificial SequenceReverse Primer for SNP ID 99 198 ccatccagac tgggtcagtg 20 199 19 DNAArtificial Sequence Description of Artificial Sequence Forward Primerfor SNP ID 100 199 ctccatgtgg gaatcggtg 19 200 20 DNA ArtificialSequence Description of Artificial Sequence Reverse Primer for SNP ID100 200 tcaaggcaca tcacatggtc 20

What is claimed is:
 1. A method for SNP typing which comprises the stepsof: simultaneously amplifying a plurality of nucleotide sequencescomprising at least one or more sites of single nucleotide polymorphismusing genomic DNA and a plurality of primer pairs; and typing fordistinguishing the site(s) of single nucleotide polymorphism ofnucleotides contained in a plurality of nucleotide sequences amplifiedin the above amplification step using the amplified nucleotidesequences.
 2. The method for SNP typing according to claim 1, whereinsaid step of amplifying employs the polymerase chain reaction using ahot start method.
 3. The method for SNP typing according to claim 1,wherein said step of amplifying employs 50 pairs or more primers.
 4. Themethod for SNP typing according to claim 1, wherein said step of typingemploys an Invader assay or a TaqMan PCR method.
 5. The method for SNPtyping according to claim 1, wherein amplification is carried out from10 ng to 40 ng of genomic DNA.